細胞激素及周邊血球細胞之早期基因表現對C型肝炎病人於干擾素合併雷巴威林療效關聯性之研究

Analysis for the cytokine and gene expression of peripheral blood mononuclear cells in the chronic hepatitis C patients during peginterferon/ribavirin combined therapy

呂明穎(Ming-Ying Lu)

指導教授 ： 余明隆 ； 張基隆

高雄醫學大學/醫學院/醫學研究所博士班/博士(2016年)

摘要

背景
C型肝炎可於週邊血液單核球細胞內複製，週邊血液單核球細胞可以產生干擾素對抗病毒感染；此外，造成慢性C型肝炎的原因已知與細胞激素表現量失衡有關。本研究旨在探討血清中細胞激素的表現及週邊血液單核細胞早期基因的表現與慢性C型肝炎患者接受干擾素合併雷巴威林療效的關聯性。

方法
研究一:細胞激素與C型肝炎的關聯性
92名慢性C型肝炎患者依據C型肝炎的血清型分別接受24週或48週的干擾素合併雷巴威林治療，以是否達成持續性病毒反應及早期病毒反應作為評估療效的指標，持續性病毒反應定義為療程結束後24週血中無法偵測到HCV病毒核酸，早期病毒反應定義為治療12週血中無法偵測到HCV病毒核酸；以免疫螢光分析的方法檢測治療期間不同時間點的血清中細胞激素表現量，檢測的細胞激素包括(1)第一型輔助T細胞媒介的細胞激素: interferon-γ, interleukin-2, TNF-α; (2)第二型輔助T細胞媒介的細胞激素: interleukin-4, interleukin-5, interleukin-6, interleukin-10; (3)免疫調控細胞激素: interleukin-1β, interleukin-8, interleukin-12。

研究二: 週邊血液單核細胞早期基因表現與C型肝炎的關聯性
27名血清型1b的慢性C型肝炎患者接受48週的干擾素合併雷巴威林治療，分別收集患者治療前、第一週及第四週的週邊血液單核細胞，先利用DNA微陣列的方法來篩選與持續性病毒反應有關的基因(n=7)，接著再以即時聚合酶鏈鎖反應來確認基因的表現量(n=20)。

結果
研究一:細胞激素與C型肝炎的關聯性
結果發現不論是特定時間點的濃度或是治療期間的interferon-γ總表現量，干擾素合併雷巴威林治療無效患者的表現量顯著高於治療成功者，多因子邏輯回歸分析發現interferon-γ為一獨立的持續性病毒反應預測因子; 次族群分析發現: 於IL28B rs8099917 基因型TT的患者，或是無法達成快速病毒反應的次族群，無法達成持續性病毒反應者治療期間interferon-γ表現量顯著高於具有持續性病毒反應者。

研究二: 週邊血液單核細胞早期基因表現與C型肝炎的關聯性
結果發現第一週及第四週時分別有13個基因及24個基因，於具有持續性病毒反應的患者基因表現量顯著高於無持續性病毒反應的患者，其中有8個基因同時在一週及第四週皆有表現；以這8個基因(RSADx, LOCxxxxx, HERCx, HERCx, IFIxx, SERPINGx, IFITMx, DDXxx)建立了一個干擾素合併雷巴威林療效的預測模式，該模式可於療程第一週時準確預測血清型1b的慢性C型肝炎患者的療效(持續性病毒反應ROC曲線下面積=0.89, p=0.007; 早期病毒反應ROC曲線下面積=0.95, p=0.003)，此預測模式特別適用於IL28B rs8099917基因型TT的患者(持續性病毒反應ROC曲線下面積=0.89, p=0.002; 早期病毒反應ROC曲線下面積=1.00, p=0.008)；以接受者操作特性曲線分析證明該基因預測模式的曲線下面積優於傳統的預測因子(包括快速病毒反應、治療前病毒量及IL28B基因型)。

結論
干擾素合併雷巴威林治療期間，若血清中有較高的interferon-γ表現量與無法達成持續性病毒反應具有顯著的相關性，特別是那些無法達到快速病毒反應或是帶有IL28B適治基因型(TT)的患者；本研究亦建立一個以檢測週邊血液單核細胞基因的預測模式，可於療程第一週時準確預估干擾素合併雷巴威林的療效。

關鍵字

C型肝炎 ； 干擾素 ； 持續病毒反應

並列摘要

Background
Hepatitis C virus (HCV) can replicate in peripheral blood mononuclear cells (PBMC), where is a potent producer of interferon in response to virus infection. Chronic hepatitis C virus infection is associated with cytokine imbalance. Cytokine dynamics and gene expression of PBMC may have an impact on the treatment efficacy of peginterferon/ribavirin for HCV patients. This study was aimed to analyze the dynamic cytokine and gene expression of PBMC in chronic hepatitis C patients during peginterferon/ribavirin therapy.

Materials and Methods
Study 1: Cytokines vs. HCV
Ninety-two treatment-naive chronic hepatitis C patients were treated with 24 or 48 weeks of peginterferon/ribavirin therapy based on the HCV genotypes. Sustained virologic response (SVR) is defined as undetectable HCV RNA throughout a 24-week post-treatment follow-up period. Early virologic response (cEVR) is defined as absence of HCV RNA at week 12 after treatment. Dynamic serum levels of the following cytokines: (1) Th1-mediated cytokines: IFN-γ, interleukin-2, and TNF-alpha; (2) Th2-mediated cytokines: interleukin-4, interleukin-5, interleukin-6, and interleukin-10 and (3) immuno-modulatory cytokines: interleukin-1β, interleukin-8, and interleukin-12 were determined by Fluorescent Bead immunoassay.

Study 2. PBMC gene expression vs. HCV
We collected the PBMCs at baseline, 1st, and 4th week from 27 chronic HCV-1 patients treated with 48-week peginterferon/ribavirin regimen. Affymetrix microarray (n=7) was used to identify differential expressed genes between SVR and non-SVR, which was further validated by quantitative polymerase chain reaction (n=20).

Results
Study 1. Cytokines vs. HCV
Not only elevated IFN-γ concentrations at specific time points but also the total IFN-γ amount was strongly associated with non-response in peginterferon/ribavirin therapy. IFN-γ levels could serve as an independent predictor for SVR analyzed by multivariate logistic regression test. The accuracy of discriminating responders from non-responders was acceptable when IFN-γ cut-off levels were set at 180, 120, and 40 pg/ml at the 4th week, 12th week, and end-of-treatment of therapy, respectively. Elevated on-treatment IFN-γ concentration was significantly associated with treatment failure among interleukin-28B rs8099917TT carriers and those patients failed to achieve rapid virologic response

Study 2. PBMC gene expression vs. HCV
Thirteen genes at week 1 and 24 genes at week 4 were differentially expressed in SVR group compared with non-SVR group. We selected 8 target genes (RSADx, LOCxxxxx, HERCx, HERCx, IFIxx, SERPINGx, IFITMx, and DDXxx) at week 1 as the major components of a scoring method to predict the treatment outcome of HCV-1 patients. This predictive model can reliably stratify the responders and non-responders at week 1 (AUC=0.89, p=0.007 for SVR; AUC=0.95, p=0.003 for cEVR), especially among the patients carrying the favorable IL28B rs8099917 TT genotypes (AUC=0.89, p=0.002 for SVR; AUC=1.0, p=0.008 for cEVR). The performance of this predictive model was superior to the traditional predictors, including rapid virologic response, viral load and IL28B genotypes.

Conclusion
Elevated on-treatment IFN-γ concentration was significantly associated with treatment failure among interleukin-28B rs8099917TT carriers and those patients failed to achieve rapid virologic response. Week 1 gene score can be used to predict the treatment efficacy of peginterferon/ ribavirin therapy for HCV-1 patients.

並列關鍵字

Hepatitis C ； interferon ； sustained virologic response

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